Borah B.JCHI-KUANG SUN2023-06-092023-06-09202226661667https://www.scopus.com/inward/record.uri?eid=2-s2.0-85128657230&doi=10.1016%2fj.xpro.2022.101330&partnerID=40&md5=6e3761ab9fa379c276c84169f45135e5https://scholars.lib.ntu.edu.tw/handle/123456789/632129A resonant-scanning multiphoton optical microscope (MPM) with a millimeter-scale field-of-view (FOV) often encounters a poor Nyquist figure-of-merit (NFOM), leading to an aliasing effect owing to limited effective voxel-sampling rate. In this protocol, we provide a design guideline to enable high-NFOM MPM imaging while simultaneously securing a large FOV/digital-resolution ratio and a fast resonant raster-scanning speed. We further provide a free version of our custom acquisition software to assist with a smooth and easy construction process. For complete details on the use and execution of this protocol, please refer to Borah et al. (2021). © 2022 The AuthorsBiotechnology and bioengineering; Cell Biology; Microscopy[SDGs]SDG1[SDGs]SDG3multiphoton microscopy; procedures; scintiscanning; software; Microscopy, Fluorescence, Multiphoton; Radionuclide Imaging; Softwarejournal article10.1016/j.xpro.2022.101330354968042-s2.0-85128657230